Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and has the fourth highest mortality rate. It is an especially major problem among Asian and African populations. Unlikely patients with other cancers, such as lung cancer and breast cancers, more than 95% of HCC patients die within five years of being diagnosed with HCC.
Although HCC is the subject of continuing investigation and its symptoms are well known, early-stage diagnosis of this disease remains difficult and the survival rate after diagnosis is very low (3%-5%).
In addition to tissue biopsies, which are used for diagnosing HCC by computed tomography (CT) and magnetic resonance imaging (MRI), bodily fluids, such as plasma, provide a clinical sample that allows for the simultaneous measurement of proteins to determine the possible presence of HCC.
To date, the biomarkers alpha-fetoprotein (AFP), des-gamma-carboxyprothrombin (DCP), glypican-3 (GPC3), alpha-1-fucosidase and transforming growth factor-b1 have been used, alone or in combination, for the clinical screening of HCC patients. Although these biomarkers are useful for the detection of HCC, they suffer from poor sensitivity and/or specificity. For example, alpha-fetoprotein, which has been used as a serum marker for HCC for many years, has low sensitivity (39%-65%) and moderate specificity (76%-94%). Thus, there is an urgent need for a new class of biomarkers with enhanced sensitivity and specificity, and which are capable of diagnosing HCC at an early stage.
The inventors of the present invention have discovered an N-linked glycosylated protein that is a specific biomarker for HCC. This protein, human liver carboxylesterase 1 (hCE1), was identified by 2-dimensional gel electrophoresis (2-D DIGE) and nano-liquid chromatography mass spectrometry (Nano-LC-MS), and found to be differentially expressed in clinical plasma samples obtained from healthy volunteers and HCC patients.
The candidate HCC biomarker protein, hCE1, which is expressed mainly in the liver, is known to be expressed at lower levels in HCC tissues relative to normal liver tissue. However, there are no reports on the direct detection of secreted hCE1 protein in human bodily fluid (i.e., plasma). Consistent with this, searches of the public plasma protein database (www.plasmaproteomedatabase.org) by the inventors have failed to uncover evidence for hCE1 in plasma.
Previous studies related to hCE1 in human plasma have used enzymatic assays employing the non-specific substrates, triolein or p-nitrophenyl acetate, to measure the activity of hCE1 or its isoforms in samples purified by sepharose-affinity chromatography. However, it has recently been reported that this hydrolytic activity is likely attributable to the esterase activity of butyrylcholinesterase, paraoxonase and albumin, which are also present in human plasma. This interpretation is supported by the observation that the specific hCE1 inhibitor, bis-p-nitrophenylphosphate (BNPP), failed to confirm the presence of hCE1 in human plasma.
In this context, the inventors have used proteomics techniques to detect hCE1 in both liver tissue and plasma. Using immunoprecipitation and the proteomics techniques, 2-D DIGE and Nano-LC-MS/MS system, the inventors have demonstrated that hCE1 is present in the plasma of both healthy volunteers and patients with HCC, and provided evidence that hCE1 represents a novel HCC biomarker.